DNA Methylation in Mammalian Genomes
The only established post-synthetic modification of DNA in higher animal and plant genomes is methylation of the 5′ position of cytosine. The proportion of cytosines which are methylated can vary from a few percent in some animal genomes (1) to 30% in some plant genomes (2). Much of this metlhylation is found at CpG sites where the symmetrically positioned cytosines on each strand are methylated. In plant genomes, similar symmetrical methylation of cytosines at CpNpG (where N can be any base) is also common (3). Such sites of methylation have also been identified at low frequency in mammalian DNA (4).
Methylation patterns are heritable as the methylase enzyme recognises as a substrate, sites where a CpG dinucleotide is methylated on one strand but the corresponding C on the other strand is unmethylated, and proceeds to methylate it (5, 6). Fully unmethylated sites do not normally act as substrates for the enzyme and hence remain unmethylated through successive cell divisions. Thus, in the absence of errors or specific intervening events, the methylase enzyme enables the stable heritability of methylation patterns.
Extensive studies of gene expression in vertebrates have shown a strong correlation between methylation of regulatory regions of genes and their lack of expression (7). Most of such studies have examined only a limited number of restriction enzyme sites using enzymes which fail to cut if their target sites are methylated. A far more limited number have been examined at all cytosine bases using genomic sequencing methods (8, 9).
Bisulphite Conversion Of DNA
Treatment of single-stranded DNA with high concentrations of bisulphite followed by alkali leads to the selective deamination of cytosine, converting it to uracil (10, 11). By contrast, 5-methyl cytosines (5meC) are resistant to this chemical deamination. When bisulphite-treated DNA is copied by DNA polymerases, the uracils are read as if they were thymines and an adenine nucleotide incorporated, while 5meC is still read as a cytosine (a G being incorporated opposite). Thus, after a region of sequence is amplified by polymerase chain reaction (PCR), cytosines in the sequence which were methylated in the original DNA will be read as cytosines while unmethylated cytosines will be read as thymines (12, 13).
PCR Amplification of Methylated and Unmethylated DNA
In order to amplify bisulphite-treated DNA, primers are designed to anneal to the sequence produced after bisulphite treatment of the DNA. Since cytosines are converted to uracils, the base in the annealing primer will be an adenine rather than a guanine for the non-converted cytosine. Similarly, for the other primer of the pair, thymines replace cytosines. To permit quantification of levels of methylation in the target DNA, primers are normally chosen to avoid sites which may or may not be methylated (particularly CpG sites) and so may contain either a 5meC or a uracil after bisulphite treatment. Use of such non-selective primers allows both methylated and unmethylated DNAs to be amplified by PCR providing for quantification of the level of methylation in the starting DNA population. The PCR-amplified DNA can be cut with an informative restriction enzyme, can be sequenced directly to provide an average measure of the proportion of methylation at any position or molecules may be cloned and sequenced (each clone will be derived from amplification of an individual strand in the initial DNA). Such studies have indicated that, while a population of molecules may conform to an overall pattern of methylation, not all molecules will be identical and methylation may be found on only a fraction of molecules at some sites (13, 16).
Selective Amplification of Methylated DNA
Recently Herman et al. (14) described a variation of the bisulphite sequencing procedure to make it selective for the amplification of only methylated DNA. In this work, PCR primers were used which were designed to discriminate between the sequences produced after bisulphite-treatment of methylated and non-methylated target DNAs. Thus, cytosines which formed part of a CpG site would not be bisulphite converted and would remain as cytosines in the methylated DNA but would be converted to uracils in the unmethylated target DNA. Primers utilising these differences were designed and used for the amplification of methylated DNA sequences from four tumour suppressor genes, p16, p15, E-cadherin and von Hippel-Lindau.
Methylation of the GLUTATHIONE-S-TRANSFERASE Pi Gene in Prostate Cancer
Lee et al. (15) (U.S. Pat. No. 5,552,277 and International Patent Application No PCT/US95/09050) demonstrated that expression of the glutathione-S-transferase (GST) Pi gene is lost in nearly all cases of prostate cancer. They further showed that in twenty cases examined, using Southern blotting, that this loss of expression was accompanied by methylation at a specific restriction enzyme site (BssHII) in the promoter region of the gene. This methylation was not seen in normal prostate tissue or in a number of other normal tissues examined. In examining a prostate cancer cell line in which the GST-Pi gene is inactive, they also identified methylation at two other restriction enzyme sites, NotI and SacII in the promoter region of the gene. Digestion of cell line DNAs with the enzymes MspI and HpaII, indicated that the correlation of DNA methylation with lack of expression was not maintained for these sites which were largely located downstream of the transcription start site. The nature of the data makes it difficult to reach conclusions on the methylation status of individual MspI/HpaII sites. However, Lee et al. (18) were able to show that following HpaII digestion (which will cut at all unmethylated HpaII sites), a region of DNA containing twelve HpaII recognition sites could be amplified by PCR from tumour DNA, but not from normal prostate or leukocyte DNA. This indicates that some DNA molecules in prostate cancer are methylated at all these HpaII sites, while DNAs from normal prostate and leukocyte DNA must contain at least one of these sites unmethylated (as a single cut will render the region incapable of being amplified by PCR).
The present inventors have identified and developed an alternative method for detecting sites of methylation present in DNA from prostate cancer tissue but not present in DNA from normal tissue. The method relies on selective amplification of a target region of the GST-Pi gene but does not require prior restriction with an informative restriction enzyme.